1. Field of the Invention
The present invention relates to an enzymatic assay method of a biological substance producing ammonia as the reaction product, wherein ammonia present in a sample of the biological substance is preliminarily consumed so as to accurately assay such substance, and also relates to an ammonia elimination reagent for enzymatic assay method using a thermostable isocitrate dehydrogenase as a coupling enzyme, the dehydrogenase being preferred for a reagent in solution and having excellent stability at alkaline pHs.
2. Description of the Prior Art
Generally, detection of substances in urine, blood serum and the like, including urea, creatinine, creatine, guanine and adenosine is routinely conducted. Also, the activities of various enzymes in relation with these substances are assayed. For the detection of such substances and the enzymatic reactions thereof, ammonia is initially generated, and the resulting ammonia is then converted into glutamic acid via glutamate dehydrogenase (abbreviated as "GLD" hereinafter) to determine at 340 nm the reduction of NAD(P)H via the coupled reaction: the reduction-type nicotine amide adenine dinucleotide (phosphate) NAD(P)H!.fwdarw.nicotine amide adenine dinucleotide (phosphate) NAD(P).sup.+ !.
Because ammonia is measured as a reaction product in the reaction system, however, ammonia originally present in a sample is also measured and included in the measured value. Therefore, it has been difficult to accurately assay such substances. Thus, ammonia present in a sample should be pretreated with 2-oxoglutaric acid via GLD to be converted to glutamic acid. Because the reaction system ammonia.fwdarw.glutamic acid involves the change of NAD(P)H.fwdarw.NAD(P).sup.+, the generated NAD(P).sup.+ should be again converted into NAD(P)H through the reverse reaction NAD(P).sup.+ .fwdarw.NAD(P)H. Then, a coupled reaction can be induced by isocitrate dehydrogenase using as the substrate isocitric acid, together with metal ions such as magnesium ion or manganese ion. The reaction scheme is shown in FIG. 1.